Recombinant Differential Anchorage Probes that Tower over the Spatial Dimension of Intracellular Signals for High Content Screening and Analysis Laura Schembri, † Marion Zanese, ‡ Gaelle Depierre-Plinet, ‡ Muriel Petit, ‡ Assia Elkaoukabi-Chaibi, ‡ Loic Tauzin, ‡ Cristina Florean, †,§ Lydia Lartigue, † Chantal Medina, † Christophe Rey, † Francis Belloc, | Josy Reiffers, † Franc ¸ ois Ichas, †,‡ and Francesca De Giorgi* ,†,‡ INSERM U916, Institut Bergonie ´ , Universite ´ Victor Segalen, 229 cours de l’Argonne, 33076 Bordeaux, France, FLUOFARMA, 2 rue Robert Escarpit, 33600 Pessac, France, Dipartimento delle Scienze Biomediche Sperimentali, Universita ` di Padova, 35121 Padova, Italia, and CHU de Bordeaux, Ho ˆpital du Haut Le ´ve ˆ que, 33600 Pessac, France Recombinant fluorescent probes allow the detection of molecular events inside living cells. Many of them exploit the intracellular space to provide positional signals and, thus, require detection by single cell imaging. We describe here a novel strategy based on probes capable of encoding the spatial dimension of intracellular signals into “all-or- none” fluorescence intensity changes (differential anchor- age probes, DAPs). The resulting signals can be acquired in single cells at high throughput by automated flow cytometry, (i) bypassing image acquisition and analysis, (ii) providing a direct quantitative readout, and (iii) allowing the exploration of large experimental series. We illustrate our purpose with DAPs for Bax and the effector caspases 3 and 7, which are keys players in apoptotic cell death, and show applications in basic research, high content multiplexed library screening, compound char- acterization, and drug profiling. Fluorescent-protein biosensors are useful tools for monitoring molecular events in live cells, such as protein translocation, enzyme activation, protein-protein interaction, and the operation of second messengers. 1,2 The generic design of fluorescent biosensors is based on the development of chimeric constructs capable of undergoing conditional changes in spectra or intensity that report the event of interest. This is generally obtained by genetic modification of the emitting protein or by the construction of fluorescence resonance energy transfer (FRET) probes. 1 However, spectral-based signals can be difficult to acquire, and this often restricts the application of these probes to single cell and single parameter studies in basic research. Recently, the interest for fluorescent protein biosensors propagated to the drug discovery field with the emergence of high- throughput cell imaging platforms 3 designed to screen large libraries of compounds or of targeted siRNAs. A new generation of positional probes based on changes in intracellular distribution was developed and implemented in cell-based high-content screening (HCS) assays running on these platforms. 3 However, this technology is objectively limited by the need of specific equipment for high-resolution image acquisition and analysis that significantly complicates the screen and exponentially increases the equipment costs as well as the amount of image data to process. 4,5 In this study, we describe an alternative approach allowing one to perform HCS assays by flow cytometry with a new class of simple fluorescent probes capable of converting positional signals into intensity responses. This method is based on the differential retention of a fluorescent recombinant probe following plasma membrane permeabilization (coined “differential anchorage probes”, DAPs) and is capable of detecting a variety of molecular events such as proteolytic activities and changes in intracellular com- partmentalization. We present here two specific DAPs targeting key molecular steps in apoptotic pathway: namely, Bax activation and caspase 3 or 7 activity. With these probes, we successfully quantified the target molecular events at high throughput and multiplexed their detection with other parameters like cell cycle status, proliferation index, or cytolysis. Our results show that DAPs constitute a robust alternative tool for quantitative and high content identification/ characterization of molecular targets, as well as for drug discovery. EXPERIMENTAL SECTION Plasmids. The cDNA encoding for GFP-Bax was a gift of R.J. Youle. The cDNA encoding the mom-C3/7 DAP was generated by insertion of the DEVD coding sequence by site-directed mutagenesis on the GFP-RR cDNA (kindly provided by Nica Borgese) with a Quick Change Site-directed mutagenesis kit (Stratagene). The DEVD -mom TMD sequence was cloned in pCopGreen, pTURBOGFP, and pHcRed-tandem plasmids (Evro- * Corresponding author. Address: INSERM U916, Institut Bergonie ´ , 229 cours de l’Argonne, 33076 Bordeaux, France. Tel: +33 (0)556330433. Fax: +33 (0)556333206. E-mail: francesca.degiorgi-ichas@inserm.fr. † Universite ´ Victor Segalen. ‡ FLUOFARMA. § Universita ` di Padova. | Ho ˆpital du Haut Le ´ve ˆque. (1) Zhang, J.; Campbell, R. E.; Ting, A. Y.; Tsien, R. Y. Nat. Rev. Mol. Cell. Biol. 2002, 3, 906–918. (2) Giepmans, B. N. G.; Adams, S. R.; Ellisman, M. H.; Tsien, R. Y. Science 2006, 312, 217–224. (3) Lang, P.; Yeow, K.; Nichols, A.; Scheer, A. Nat. Rev. Drug Discovery 2006, 5, 343–356. (4) Baatz, M.; Arini, N.; Scha¨pe, A.; Binnig, G.; Linssen, B. Cytometry, Part A 2006, 69, 652–658. (5) Gribbon, P.; Sewing, A. Drug Discovery Today 2003, 8, 1035–1043. Anal. Chem. 2009, 81, 9590–9598 10.1021/ac9015227 CCC: $40.75  2009 American Chemical Society 9590 Analytical Chemistry, Vol. 81, No. 23, December 1, 2009 Published on Web 10/29/2009